Evidence for involvement of the alcohol consumption WDPCP gene in lipid metabolism, and liver cirrhosis

Biological pathways between alcohol consumption and alcohol liver disease (ALD) are not fully understood. We selected genes with known effect on (1) alcohol consumption, (2) liver function, and (3) gene expression. Expression of the orthologs of these genes in Caenorhabditis elegans and Drosophila melanogaster was suppressed using mutations and/or RNA interference (RNAi). In humans, association analysis, pathway analysis, and Mendelian randomization analysis were performed to identify metabolic changes due to alcohol consumption. In C. elegans, we found a reduction in locomotion rate after exposure to ethanol for RNAi knockdown of ACTR1B and MAPT. In Drosophila, we observed (1) a change in sedative effect of ethanol for RNAi knockdown of WDPCP, TENM2, GPN1, ARPC1B, and SCN8A, (2) a reduction in ethanol consumption for RNAi knockdown of TENM2, (3) a reduction in triradylglycerols (TAG) levels for RNAi knockdown of WDPCP, TENM2, and GPN1. In human, we observed (1) a link between alcohol consumption and several metabolites including TAG, (2) an enrichment of the candidate (alcohol-associated) metabolites within the linoleic acid (LNA) and alpha-linolenic acid (ALA) metabolism pathways, (3) a causal link between gene expression of WDPCP to liver fibrosis and liver cirrhosis. Our results imply that WDPCP might be involved in ALD.

Advances in omics such as genomics and metabolomics within the last two decades have resulted in a boost in our understanding of the mechanism of diseases through agnostic approaches such as genome-wide association studies (GWAS) that revealed numerous genetic loci linked to complex diseases.Recently, we have applied GWAS to identify genetic variants in the form of single nucleotide polymorphisms (SNPs) that are associated with alcohol consumption 3 as well as with circulating liver enzymes 4 in the European populations.Some of the identified alcohol genes (e.g., ADH, KLB, DRD2) have been investigated for a better understanding of their involvement in alcohol consumption and health consequences such as hepatic lipogenesis.However, the biological effect of most of the identified alcohol-associated genes remains to be elucidated.
In this study, we aimed to shed light on the biological pathways and molecular changes linking alcohol consumption and liver pathologies.We first investigated molecular consequences of alcohol consumption and the pathways involved at metabolic level using omics approaches.Subsequently in search for common pathways between alcohol consumption and liver pathologies, we identified candidate genes with effect on both alcohol consumption and liver function and then investigated the biological effect of candidate genes in model organism i.e., ethanol-exposed C. elegans and Drosophila to generate knowledge that could ultimately be used to better understand alcohol related behavior and hepatic lipogenesis.We finally returned to use data from humans to further validate the most plausible candidate genes with evidence of potential involvement in lipogenesis.

Methods Population
In the current study, we followed a multi-stage approach using population-based studies and model organisms to better understand pathways involved in alcohol consumption and its health consequences (Fig. 1).We used data from the Airwave Health Monitoring Study 5 , an occupational cohort of 53,116 police officers and staff ages 18 years and over across the UK (Supplementary Table 1).The Airwave Health Monitoring Study was approved by the UK National Research Ethics Service (NRES) North West-Haydock Regional Ethics Committee (REC reference: NRES/19/NW/0054; IRAS ID: 259978).The North-West Haydock REC approved the study protocol and all study documentation, and prior individual informed consent was obtained from each of the study participants.All methods were carried out in accordance with relevant guidelines and regulations.Participants were informed about the study and provided informed consent.Detailed information about the Airwave population, metabolic assays, data processing, metabolite annotation as well as genotyping and imputation is included in the "Supplementary Methods".

Alcohol consumption
Alcohol consumption during the last seven days was assessed through a self-reported questionnaire in the Airwave study.The participants were asked to quantify (1) the number of glasses (small/125 ml) of red wine, white wine/champagne, fortified wine (includes sherry, port and vermouth), (2) the number of pub measures of spirits/liqueurs (includes whisky, gin, rum, vodka and brandy), and (3) the number of pints of beer or cider (include bitter, lager, stout, ale and Guinness) consumed in the last seven days.We considered the density of alcohol to be 0.79 g/ml at room temperature and calculated the total amount of alcohol consumed by summing up the grams of alcohol consumed from each category of alcoholic drink.www.nature.com/scientificreports/

Analysis of the metabolome
We analyzed the association between alcohol consumption and circulating metabolites within the Airwave sample to identify candidate metabolites associated with alcohol consumption within the Airwave sample (alcoholassociated metabolites).Metabolomics features (n = 4092) were obtained from data acquired by the National Phenome Centre (NPC) using liquid-chromatography/mass spectrometry), covering a wide range of hydrophilic and lipid metabolite classes.We excluded unannotated features leaving 986 annotated metabolites for the analysis and tested the association of these metabolites with alcohol consumption.Subsequently, to assess if the resulting alcohol-associated metabolites were additionally involved in known metabolic pathways, we performed pathway enrichment analysis using all annotated alcohol-associated metabolomics features.To this end, Kyoto Encyclopedia of Genes and Genomes (KEGG) 6 and The Small Molecule Pathway Database (SMPDB) 7 were used and the statistical significance level was claimed using a P-value calculated using false discovery rate (Pfdr).

Genotyping and imputation
In the Airwave study 5 , genotyping was conducted using Illumina Infinium HumanExome-12v1-1 BeadChip Array.QC steps were performed to remove any samples with high missingness (> 3%) or outlier heterozygosity rates (> 3SD from the batch mean), duplicates (ID-based or genotype-based) or presented with high degree of relatedness.Markers with genotype call rate < 98%, significant deviation from Hardy-Weinberg equilibrium (P < 1 × 10 -5 ), and low MAF (< 1%) were removed.Genotype data was imputed with 1000 Genomes phase 3 reference panel.

GWAS on metabolomics
We performed genome-wide association studies (GWAS) in the Airwave Study to obtain genetic association estimates for alcohol-related SNPs and selected metabolomic features.Untargeted Mass-Spectrometry (MS) was performed in heparin plasma samples of the Airwave Study, producing three separate datasets, namely hydrophilic interaction liquid chromatography (HILIC) positive (HPOS), lipid positive (LPOS), and lipid negative (LNEG).For each metabolomic dataset, we used principal component analysis (PCA) to identify outlier samples and exclude them.The data was residualized using the first ten principal components to account for population stratification.We transformed the data into z-scores, performed individually for each metabolomic feature, using median and median absolute deviations (MAD).Values that were more than 5 MAD from the median were removed and imputed using k-nearest neighbors' imputation 8 .GWAS were conducted for each metabolomic feature with adjustment for age and sex (N = 1970).Due to the high number of metabolomic features, GWAS were performed using the high-dimensional association analyses (HASE) framework, which applies matrix operation and removes redundant calculation in high-dimensional association analyses to improve computational efficiency 9 .We used the results from association analyses between the alcohol-associated metabolites and the known alcohol-associated SNPs to further perform a causal inference analysis on these results using the inverse variance weighted two-sample Mendelian randomization (MR) analysis 10 .The aim of the MR analysis was to identify changes in circulating metabolites caused by alcohol consumption.MR is a causal assessment method used in observational studies to mimic randomized controlled trials (RCTs) by taking advantage of the random assortment of alleles at conception.MR uses instrumental variables (i.e., genotype status), that are robustly associated with an exposure of interest, as a natural randomization tool occurring at conception 11 .The detailed methods for selection of alcohol consumption instruments have been described previously 10,12 .Briefly, we obtained genetic association statistics (β values) for 105 alcohol-associated SNPs 3,13 from Liu and colleagues 13 .Clumping and removing weak instruments as described previously 10,12 ensured the most robust instruments were used for the MR analysis (n = 33).

Gene selection for model organisms
We selected a list of 105 alcohol-related SNPs from recently conducted GWAS of alcohol consumption 3,13 .SNPs were selected if they presented a P-value lower than a GWAS significance threshold of 5 × 10 −8 in their association with alcohol consumption.As we were interested in finding pathways involved in hepatic lipogenesis as a result of alcohol consumption, we sought candidate SNPs that showed strong effect with both alcohol consumption and liver function, using our recently published GWAS of circulating liver enzymes 4 .To account for multiple testing, a corrected P-value threshold of < 0.00048 was used for the association with liver enzymes.This P-value threshold corresponds to a nominal P-value (0.05) that has been adjusted for the number of alcohol-related SNPs (n = 105) using the Bonferroni method 14 .SNPs (n = 43; Supplementary Table 2) that were associated with at least one of the three liver enzymes alanine transaminase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) were shortlisted to assess their link to gene expression using the GTEx database 15 .We eventually selected SNPs (n = 24) that demonstrated evidence of a statistically significant effect on gene expression of their nearest genes (a cis-eQTL effect; Table 1).

Ortholog selection
The 24 SNPs with cis-eQTL effect with their annotated genes were matched to their orthologs in Drosophila using FlyBase (DIOPT online tool version 8.5/9.0;beta; http:// www.flyrn ai.org/ diopt).To ensure the selection of the most credible orthologs, we used scores calculated in FlyBase.This database provides a number of approaches that support the gene-pair relationship out of a total number of tools that computed relationships between Homo sapiens and Drosophila.Genes with a score of > 12 were shortlisted for further analysis in Drosophila.Nineteen Drosophila orthologs were identified of which eight had a score ≥ 12 including ARPC1B (arpc1), ACTR1B (arp1), GPN1 (CG3704), WDPCP (frz), MLXIPL (mondo), SLC4A8 (ndae1), SCN8A (para), and TENM2 (Ten-m).

Drosophila
Genetics and Drosophila melanogaster strains All Drosophila stocks and crosses were maintained on standard cornmeal agar media at 25 °C on 12/12 h light/ dark cycles.The following strains were used as positive control: w berlin ; hppy  ; + (hppy mutant) and w berlin ; hppy  ; hppy III (hppy mutant with a rescue genomic construct) (gifts from Prof. Ulrike Heberlein, Janelia Research Campus, Virginia, USA), RNAi lines were obtained from Bloomington Drosophila Stock Center (gene CG3704 (BDSC no: 55294), Frz (BDSC no: 55649), Mondo (BDSC no: 27059), Arpc1 (BDSC no: 31246), Para (BDSC no: 33923) and TenM (BDSC no: 29390)). All lnes used were backcrossed to w 1118 or [v] w1118 (RNAi lines).The expression of all RNAi constructs was driven by the ubiquitous driver, daGal4, which drives expression throughout development from embryonic to adult stage in all tissues.All the experiments on adult flies were performed using males.

Drosophila ethanol consumption assay
The CApillary FEeder (CAFE) assay 19 was used to measure ethanol consumption.Eight male flies were placed into an experimental vial (8 cm height, 3.3 cm diameter) containing 6 microcapillary tubes (BRAND® disposable BLAUBRAND® micropipettes, intraMark, BR708707, with 1 µl marks), each containing 5 µl of liquid food.Liquid food was prepared by dissolving 50 mg of yeast granules in 1 ml of boiling water by vortexing, followed by brief centrifugation.Then, 40 mg of sucrose (Sigma-Aldrich, 84097) was added to 800 µl of the dissolved yeast mixture, followed by vortexing.The microcapillary tubes were filled with liquid food up to the 5 µl mark.Ethanol food consists of normal food supplemented with 15% ethanol.Each experiment consisted of 5 experimental vials per genotype with each vial containing normal food (3 capillaries) and ethanol food (3 capillaries).The flies were acclimatized in the experimental vial without any food for 2 h prior to the start of the experiment.This step was also used to incentivise the flies to eat once the food was introduced.The experimental vials were placed in a plastic box with a cover to control humidity.The flies were allowed to feed for 19 h, after which the amount consumed (in mm) was measured with a digital calliper (Dasqua Bluetooth Digital Calliper 12″/300 mm, 24108120).The total amount of food consumed was calculated using the formula:

Drosophila ethanol sedation assay
Fly sedation assay was performed as previously described 20 .Briefly, 8 flies were transferred to a 25 mm x 95 mm transparent plastic vial in between two cotton plugs.A piece of cotton plug at the base of the vial served as a stable surface to observe the flies and another plug was used to cap the vial and deliver the ethanol.500ul of 100% ethanol was added to the side of the cotton plug facing the flies.Sedation was observed manually as ST50, which is the time in minutes it takes for 50% of the flies in a sample vial to become sedated.Sedation events are recorded when the flies become inactive and lay on their backs for over 10 s.

Drosophila triradylglycerols (TAG) measurements
To assess the role of the candidate genes in lipogenesis, we assessed the effect of RNAi knockdown of the selected genes on TAG levels in Drosophila.Eight male flies of the indicated genotypes were placed into an experimental vial as described in the ethanol consumption assay, with all 6 microcapillary tubes filled with normal food (5% sucrose + 5% yeast) or ethanol food (normal food + 15% ethanol), for 2 days.TAGs were assessed through colorimetric assays using 96-well microtiter plates and an Infinite M200Pro multifunction reader (TECAN).The assays were performed as previously described 21 .Briefly, flies were homogenized in 110 µl of PBS + 0.05% Tween 20 (PBST) for 2 min on ice and immediately incubated at 70 °C for 10 min to inactivate endogenous enzymatic activity.A 35 µl fly homogenate sample and a glycerol standard (Sigma, no.G7793) were incubated together with either 35 µl of PBST (for free glycerol measurements) or 35 µl of TAG reagent (Sigma, no.T2449, for TAG measurements) at 37 °C for 60 min.After 3 min of centrifugation at full speed, 30 µl of each sample was transferred into a clear-bottom plate (two technical replicates per biological sample) together with 100 µl of free glycerol reagent (Sigma-Aldrich, F6428) and incubated at 37 °C for 5 min.TAG absorbance was divided by the protein concentration of the respective sample, which was measured by Bradford assay (Sigma-Aldrich, B6916).

C. elegans
Nematode strains and culture All C. elegans strains were cultured on nematode growth medium (NGM) agar plates at 20 °C using Escherichia coli OP50 as a food source.For the wildtype worms, Bristol N2 strain was used.Genes for which mutations were homozygous lethal (arp-1, arx-3, gop-2 and ten-1), heterozygote mutations balanced by chromosomal translocations were instead analyzed.Loss-of-function mutations were not backcrossed into Bristol N2, but instead initially screened for phenotypic differences with those differences validated by RNAi.
Food uptake (µl) = � measured distances between 3 microcapillary tubes (mm) / www.nature.com/scientificreports/Nematode RNA interference (RNAi) RNAi experiments were performed on the NL2099 rrf-3 (pk1426) strain as previously described 16,22 (Supplementary Table 3).RNAi was achieved by feeding 23 using the ORFeome based RNAi library 24 .In brief, HT115 RNAi bacterial clones were initially cultured in LB media with 100 µg/ml ampicillin and subsequently spotted in three 50 µl drops on 60 mm diameter NGM plates containing 1 mM isopropyl β-1-thiogalactopyranoside (IPTG) and 25 µg/ml carbenicillin.Plates were left to dry for 4-7 days before seeding to improve RNAi efficiency.Following seeding, five L3-L4 worms were added to each RNAi plate and cultured at 20 °C until the F1 generation reached adulthood.Ethanol experiments were performed and analyzed as described above and compared to worms fed with an empty RNAi feeding vector.

Nematode behavioural assays
All ethanol experiments were performed at 20 °C in a temperature-controlled room as previously described 16,22 .

Secondary analysis
To gain a better insight into the biological pathways involved in the link between alcohol consumption and liver damage, we used the genetic variants within the genes highlighted by our model organism experiments and performed a series of secondary analyses using human data.We explored publicly available data from the UK Biobank deposited in the Edinburgh Gene Atlas 26 using Phewas (Phenome-wide association analysis) databases to obtain association results between the genetic variants and 778 traits.We additionally used the genes highlighted by our model organism experiments to assess the causal effect of gene expression on liver conditions.Within these genes, the SNPs that have been identified to have a cis-eQTL effect within the previously published studies were selected and used as MR instrument against liver conditions within the twosampleMR package in R.

Statistical analyses
Within the Airwave study sample, we performed a linear regression to study the association of alcohol consumption with each of the metabolomic features (Metabolome-wide association study; MWAS).We adjusted the statistical analysis for age, sex, smoking status, and salary class.To account for multiple testing and the high degree of correlation in metabolomics datasets, we used a permutation-based method to estimate the significance level of the associations 27,28 .For each metabolomics platform, a P-value threshold equivalent to adjusting to a 5% Family-Wise Error Rate (i.e., Bonferroni method) was computed.A series of hypergeometric tests implemented in the R package MetaboAnalystR 29 was used for pathway enrichment analysis where an FDR threshold of 0.05 was used as a significance threshold for each of the metabolomics platforms.To obtain an estimation for the association of known alcohol SNPs with our alcohol-associated metabolites to be used in the MR analysis, we performed linear regression analysis within the Airwave sample (see "Supplementary Methods" for details of the GWAS on metabolomics).Linear analyses between SNPs and metabolites were conducted for each metabolomic feature with adjustment for age, sex, and genetic principal components within a subsample of Airwave that included participants with both genetic and metabolite data (N = 1970).In C. elegans, locomotion rate was presented normalized as a percentage of the mean thrashing rate of untreated worms measured each day.All worm data were expressed as mean ± SE with an N = 30 individual worms.Locomotion rate significance was assessed by one-way analysis of variance (ANOVA) with post-hoc Tukey test for multiple comparisons.Statistical analyses of the Drosophila experimental data were performed using GraphPad Prism (www.graph pad.com).Drosophila data were presented as the mean values, and the error bars indicate ± SD.

Secondary analysis
The Phewas analysis (Table 3) on the three genetic variants within WDPCP, TENM2, and GPN1 showed a link between rs10078588 (TENM2) and food and liquid intake (beef, oily fish, fresh fruit, bread, alcohol).rs13032049 (WDPCP) was linked to salt intake and smoking.rs2178197 (GPN1) was linked to hypertension, and hematologic traits.All three SNPs showed association with disorders of lipoprotein metabolism and other lipidaemias (ICD code E78) within the UK Biobank (Table 4).We finally performed a Mendelian randomization analysis on the expression of WDPCP gene (ENSG00000115507) and liver conditions using cis-eQTL data (Table 5) and identified a link between expression of WDPCP with liver fibrosis and cirrhosis (β = − 0.20; 95% CI − 0.39, − 0.01; P-value = 0.04) as well as liver and bile duct cancer (β = 0.0003; 95% CI 3.27 × 10 -05 , 5.85 × 10 -04 ; P-value = 0.02).We also observed a suggestive link with the changes in liver fatty acid-binding protein (Table 5).

Discussion
In this study we used data from humans, C. elegans, and Drosophila and identified a link between genes implicated in alcohol consumption and lipid metabolism.We identified that alcohol consumption changes the metabolites within linoleic acid (LNA) and alpha linolenic acid (ALA) metabolism pathway (LNA/ALA) and demonstrates causal effect on changes in several lipid metabolites.We highlighted that change of function of the genes implicated in alcohol consumption leads to changes in ethanol consumption, sedation after exposure to ethanol vapor, and changes in accumulation of fat in Drosophila as well as changes in locomotion rate after exposure to ethanol in C. elegans.Our results demonstrate that three alcohol-implicated genes namely WDPCP (frz), TENM2 (Tenm), and GPN1 (CG3704) might be involved in fat accumulation.In our study, we identified several metabolites to be causally altered due to alcohol consumption and identified the LNA/ALA pathway involved in alcohol consumption.A metabolite, eicosapentaenoic acid that is synthesized from linolenic acid, has previously been associated with altered acute behavioral responses to alcohol in C. elegans (altered locomotion) 31 , and mice 32 .In addition in humans, long-chain polyunsaturated fatty acids are shown to be associated with alcohol sensitivity 33 and genes that are essential to generate ω-3 long-chain polyunsaturated fatty acids are shown to be associated with alcohol-related phenotypes 34 .Previous studies also showed associations between lipid metabolites and alcohol consumption 35 .We previously showed alteration of high-density lipoprotein to be associated with alcohol consumption within the UK Biobank 10 .The metabolomics part of the current study is a step forward in that firstly it uses causal inference tools (MR method) to demonstrate the causality of the association between lipid metabolites specifically long chain fatty acids with alcohol consumption.Secondly, by performing a pathway analysis, we were able to pinpoint a specific LNA/ALA pathway in alcohol consumption.
In our study, we used alcohol-associated genetic variants to explore (1) the alcohol-induced biological changes in human metabolites and (2) alcohol-induced biological effect of genes annotated to alcohol-associated genetic variants in C. elegans and Drosophila melanogaster.Of the alcohol-implicated genes that we investigated in C. elegans, ACTR1B (arp-1) and MAPT (ptl-1), show significant effects on the worms' locomotion upon acute exposure to ethanol.In addition, TENM2 (Ten-m) shows significant effects on ethanol consumption in Drosophila and apart from MLXIPL (mondo), RNAi lines for all the genes investigated in Drosophila change the time to sedation from ethanol.
WDPCP which was first characterized in Drosophila 36 is known to be involved in cell polarity and ciliogenesis 37,38 .In humans, mutations in WDPCP gene cause Bardet-Biedl Syndrome 39 presenting with a variety of symptoms in different organs including obesity, blindness, and polydactyly.WDPCP gene is widely expressed known to be involved in hedgehog signaling 40 .Recently, Liu and colleagues 13 identified that a G allele in rs13032049 within the WDPCP gene was associated with an increased consumption of alcohol.The G allele in rs13032049 shows a strong association with a lower expression of the WDPCP gene.We found the SNP to be strongly associated with increased liver enzyme GGT and behavioral traits such as smoking and adding salt to food.In our Drosophila experiments, the observed effect of WDPCP (frz) knockdown on the changed TAG levels occurred under exposure to ethanol and followed similar patterns as hppy mutants.This implies that minimizing alcohol consumption could reduce fat accumulation and thus could potentially reduce the risk of hepatic lipogenesis.In our further MR analysis, we used publicly available databases derived from GWAS in human population and showed a link between the gene expression of WDPCP and liver fibrosis and liver cirrhosis.The www.nature.com/scientificreports/analysis also showed a suggestive link with the liver fatty acid binding protein that is involved in the metabolism of lipids 41 .This evidence suggests that WDPCP might be an important gene involved in the pathway between alcohol consumption, accumulation of fat and liver fibrosis.This could have public health implications in terms of the identification of high-risk groups and targeting preventive measures as well as drug development.More Table 3. Overview of the significant associations between SNPs in WDPCP, TENM2, and GPN1 with Phewas traits within the UK Biobank Edinburgh Gene Atlas.Significant associations are depicted in bold.Statistical significance level P-value = 2.14 × 10 -5 was considered equivalent to a P-value adjusted for multiple testing for the analysis of 3 SNPs and 778 phenotypes within the Gene Atlas.The associations are provided per copy of the allele that increases alcohol intake.Changes in TAG levels of Drosophila occurred with exposure to normal food rather than ethanol for RNAi knockdown of TENM2 (Ten-m) and GPN1 (CG3704) indicating that loss of function of these genes could have a direct role in the accumulation of fat in liver independent of exposure to ethanol.RNAi knockdown of both genes shows increased tolerance to the sedative effect of ethanol which could justify the effect of these genes on a more frequent alcohol consumption in humans, possibly due to alcohol tolerance.GPN1 is located on chromosome 2p23.3and the encoded protein is implicated in the regulation of TGFβ superfamily signaling 42 that is demonstrated to play a role in obesity 43 , accumulation of fat in the liver 42,44 as well as regulation of ADH1 gene that enhances alcohol-induced liver damage and lipid metabolism 45 .The existing evidence alongside our findings on the role of GPN1 in alcohol consumption and lipid metabolism in Drosophila implies that GPN1 might play a role upstream of TGFβ in the regulation of the metabolism of alcohol and lipids.Further studies are needed to highlight the relationship between GPN1 and TGFβ in alcohol consumption and alcohol-induced liver damage.
TENM2 (Ten-m) is located on chromosome 5q34, and the encoded protein is involved in cell adhesion 46 .TENM2 is found to be highly enriched in white adipocyte progenitor cells 47 .TENM2 deficiency in human fat cells leads to expression of UCP1, the primary marker of brown adipose tissue 48 .Genetic variants in TENM2 have shown to be linked to obesity 49 .Our secondary analyses confirmed an association between the genetic variant in TENM2 and excess of food and liquid intake which suggests the link between TENM2, and alcohol consumption could also be due to systematic increase in consumption of all food and beverages rather than alcohol alone.The evidence in this study alongside the existing literature highlights that the observed effect of TENM2 (Ten-m) RNAi knockdown on the changes in TAG levels in Drosophila could potentially be related to biological pathways implicated in adipose tissue rather than pure liver-related pathways.
One strength of our study is in that we performed our analyses in human and two different model organisms, allowing for a more comprehensive insight into biological mechanisms involved in the function of alcohol consumption genes under different biological scenarios.A second strength of this study is in the use of RNAi technique which provides insight into the function of genes and what biological manifestation they would have when exposed to ethanol.The third strength of our study is in the use of CAFE assay that allows for the investigation of food and alcohol consumption in Drosophila in a more controlled environment.In our CAFE assay, each experimental box per genotype contained both normal food and ethanol food (food supplemented with 15% ethanol) providing the insects with a choice.Another strength of our study is that in our TAG levels experiments, we made our conclusions based on the comparisons between flies (RNAi vs. control) that were exposed to identical food and environmental conditions which increases the robustness of our conclusions.Finally, we combined the results with human studies to get better insight into the link between alcohol consumption and lipid metabolites.
Alcohol consumption in Airwave participants was calculated based on self-reported data which could have affected the precision of the alcohol consumed due to recall bias.To reduce this limitation, the duration of recall was limited to the last 7 days in the Airwave study.We should acknowledge that the concentration of alcohol within alcoholic drinks is not standard 50 , and our calculation of alcohol consumed could be affected by these variations.Although the genetic variants used for our investigations were originally found in human studies and we also performed a metabolomics analysis between alcohol consumption and circulating metabolites in the human population, the main part of the study was performed in model organisms and the results of this study might not directly generalisable to patients and the public without performing further population studies.

Conclusion
We found that alcohol-associated genes may be involved in the metabolism of lipids.Our study highlights three genes (WDPCP, TENM2, and GPN1) that may be involved in the accumulation of lipids.Of these genes, WDPCP exhibits its effects on lipid accumulation in Drosophila with exposure to ethanol.The gene expression of WDPCP in the human population supports a link to liver fibrosis.Further studies are necessary to investigate the role of this gene in ALD.

Figure 2 .
Figure 2. Analysis of alcohol intake and sedation in adult flies.(A) Mapping Drosophila orthologues of human genes involved in ethanol consumption.Similarity of protein alignment based on the DRSC integrative ortholog prediction tool (DIOPT).(B) Analysis of ethanol intake (left column) and sedation (right column) in the Drosophila RNAi lines.The numerical values show the difference between means ± standard error of mean (RNAi-Control).Red or blue correspond to the measurements that were significantly increased or decreased, respectively (P ≤ 0.05) whereas grey indicates a significance higher than 0.05.The statistical significance was determined using an unpaired t-test.Values in parenthesis are the number of biological replicates for respectively, the control and the RNAi line.(C) Analysis of TAG levels in adult flies fed either with normal or ethanol-containing food (mean ± standard error of mean; asterisks, 2-way ANOVA with Tukey's multiple comparisons test).The number of biological replicates per experimental variable (n) is indicated in either the respective figure or figure legend.No sample was excluded from the analysis unless otherwise stated.Blinding was not performed.Normality was assessed before deciding on which parametric or non-parametric test to use for inferential statistics.Statistical significance is indicated as * for P < 0.05, ** for P < 0.01, *** for P < 0.001, **** for P < 0.0001 and NS for P ≥ 0.05.

Table 1 .
Overview of the genetic variants with effect on alcohol consumption, liver enzymes and gene expression.eQTLexpression quantitative trait loci.*AlcoholSNPs associated with liver enzymes at replication P value < 0.000476.Liver enzyme GWAS summary statistics obtained from Pazoki et al. 2021.†Normalized effect size and P-value obtained from GTEx portal.‡Orthologs were extracted from FlyBase and WormBase.We did not identify any fly or worm orthologs for NUCKS1, GCKR, BDNF, FTO, TCAP.Additionally, no worm ortholog was found for WDPCP, MSANTD1, MYBPC3, PMFBP1, TNFSF13, and ACSS1.

Table 2 .
Overview of the causal effect of alcohol on circulating metabolites using the inverse variance weighted two-sample Mendelian randomization multiple instrument method.Effect estimates and 95% CI is given for the inverse variance weighted method of Mendelian randomization.CI confidence interval, DAG diradylglycerols, TAG triradylglycerols.

Table 4 .
Overview of the association of SNPs in WDPCP, TENM2, and GPN1 with disorders of lipoprotein metabolism and other lipidaemias (ICD code E78) within the UK Biobank Edinburgh Gene Atlas.Results obtained from the Edinburgh Gene Atlas.CI confidence interval, SNP single nucleotide polymorphism, MAF minor allele frequency, HWE Hardy Weinberg Equilibrium.

Table 5 .
51erview of the significant results from inverse variance weighted Mendelian randomization analysis for the effect of gene expression of ENSG00000115507 on liver traits using the MRC IEU OpenGWAS data infrastructure51.

of SNPs Effect estimate Standard error P value
studies in vivo and in vitro are needed to focus on WDPCP and provide more details on its role in lipid metabolism and liver pathologies.